Methods for preparing particles and related compositions

ABSTRACT

Methods for preparing particles and related compositions are provided. In some embodiments, the particles include at least one polynucleotide (e.g., mRNA), and in certain embodiments, the particles may include at least one ionizable molecule (e.g., a lipid). A method for preparing a suspension including the particles may comprise one or more filtration steps. In some such embodiments, prior to or during filtration, one or more properties of the particles (e.g., surface charge) and/or one or more properties of the suspension (e.g., pH) may be altered. In some embodiments, altering one or more properties of the particles and/or suspension may improve yield, improve a characteristic of the resulting composition, and/or prevent or reduce certain problems, such as fouling during the filtration process.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 62/206,121, filed Aug. 17, 2015, thecontents of which are incorporated herein by reference in its entiretyfor all purposes.

FIELD OF INVENTION

The present embodiments relate generally to methods for preparingparticles comprising polynucleotides and related compositions.

BACKGROUND

It is of great interest in the fields of therapeutics, diagnostics,reagents, and for biological assays to be able control proteinexpression. Most methods rely upon regulation at the transcriptionallevel (e.g., from DNA to mRNA), but not at the translational level(e.g., from mRNA to protein). Although attempts have been made tocontrol protein expression on the translational level, the low levels oftranslation, the immunogenicity of the molecules, and other deliveryissues have hampered the development of mRNA as a therapeutic.

There remains a need in the art to be able to design, synthesize anddeliver a nucleic acid, e.g., a ribonucleic acid (RNA) such as amessenger RNA (mRNA) encoding a peptide or polypeptide of interestinside a cell, whether in vitro, in vivo, in-situ, or ex vivo, such asto effect physiologic outcomes which are beneficial to the cell, tissueor organ and ultimately to an organism.

SUMMARY OF THE INVENTION

Methods for preparing particles comprising polynucleotides and relatedcompositions associated therewith are provided. In some embodiments, thepreparation involves one or more filtration steps. The subject matter ofthis application involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of structures and compositions.

In some embodiments, a series of methods are provided. In oneembodiment, a method comprises changing a pH of a suspension comprisingparticles comprising mRNA and an ionizable molecule from a first pH to asecond pH, wherein the second pH is greater than a pKa of the ionizablemolecule, and filtering the suspension to produce a filtered suspensioncomprising at least a portion of the particles, wherein a coefficient ofvariation of a cross-sectional dimension of the particles in thefiltered suspension is less than or equal to about 20%.

In another embodiment, a method comprises changing a pH of a suspensioncomprising particles comprising mRNA and an ionizable molecule from afirst pH to a second pH, wherein the second pH is greater than a pKa ofthe ionizable molecule, and forming a composition comprising at least aportion of the particles, wherein a coefficient of variation of across-sectional dimension of the particles in the composition is lessthan or equal to about 20%.

In another embodiment, a method comprises changing an average zetapotential of a plurality of particles comprising mRNA in a suspensionfrom a first zeta potential to a second zeta potential, wherein thesecond zeta potential is less than the first zeta potential, andfiltering the suspension to produce a filtered suspension comprising atleast a portion of the particles, wherein a weight percentage of mRNA inthe particles in the filtered suspension is greater than or equal toabout 50% and less than or equal to about 99%.

In another embodiment, a method comprises changing an average zetapotential of a plurality of particles comprising mRNA in a suspensionfrom a first zeta potential to a second zeta potential, wherein thesecond zeta potential is less than the first zeta potential, and forminga composition comprising at least a portion of the particles, wherein aweight percentage of mRNA in the particles in the composition is greaterthan or equal to about 50% and less than or equal to about 99%.

In another embodiment, a method comprises changing an average zetapotential of a plurality of particles comprising polynucleotides havinggreater than or equal to 50 nucleotides in a suspension from a firstzeta potential to a second zeta potential, wherein the second zetapotential is less than the first zeta potential, and filtering thesuspension to produce a filtered suspension comprising at least aportion of the particles, wherein a weight percentage of mRNA in theparticles in the filtered suspension is greater than or equal to about50% and less than or equal to about 99%.

In another embodiment, a method comprises changing an average zetapotential of a plurality of particles comprising polynucleotides havinggreater than or equal to 50 nucleotides in a suspension from a firstzeta potential to a second zeta potential, wherein the second zetapotential is less than the first zeta potential, and forming acomposition comprising at least a portion of the particles, wherein aweight percentage of mRNA in the particles in the composition is greaterthan or equal to about 50% and less than or equal to about 99%.

In another embodiment, a method of filtering comprises filtering asuspension comprising particles comprising a polynucleotide and anionizable molecule having a pKa less than the pH of the suspension,wherein the filtration step comprises a concentration step and adiafiltration step, and wherein the permeate flux throughout thediafiltration step is greater than or equal to about 20 L/m²h when thetransmembrane pressure is between about 1 psi and about 20 psi.

In another set of embodiments, compositions are provided. In oneembodiment, a composition comprises a plurality of particles comprisingmRNA, wherein an average cross-sectional dimension of the particles inthe composition is less than or equal to about 150 nm, a coefficient ofvariation of a cross-sectional dimension of the particles in thecomposition is less than or equal to about 20%, and a weight percentageof mRNA in the particles is greater than or equal to about 50% and lessthan or equal to about 99%.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 is a process flow diagram showing a method of forming particlescomprising polynucleotides, according to certain embodiments;

FIG. 2 is a schematic diagram of a graph showing the magnitude of chargeof particles versus pH, according to certain embodiments; and

FIG. 3 is a schematic diagram of particles described herein, accordingto certain embodiments;

FIG. 4 shows a graph of percent encapsulation versus residence time inan acidic buffer, according to certain embodiments;

FIG. 5 shows a graph of zeta potential versus pH for various particles,according to certain embodiments;

FIG. 6A shows a graph of permeate flux versus time for suspensions withvarious pHs, according to certain embodiments;

FIG. 6B shows a graph of permeate flux at different stages of tangentialflow filtration for suspensions with various pHs, according to certainembodiments;

FIG. 7A shows the average particle size during various stages oftangential flow filtration and after a sterile filtration forsuspensions with various pHs, according to certain embodiments;

FIG. 7B shows the percent polydispersity for particles in suspensionswith various pHs during various stages of tangential flow filtration andafter a sterile filtration, according to certain embodiments.

DETAILED DESCRIPTION

Methods for preparing particles and related compositions are provided.In some embodiments, the particles include at least one polynucleotide(e.g., mRNA), and in certain embodiments, the particles may include atleast one ionizable molecule (e.g., a lipid). A method for preparing asuspension including the particles may comprise one or more filtrationsteps. In some such embodiments, prior to or during filtration, one ormore properties of the particles (e.g., surface charge) and/or one ormore properties of the suspension (e.g., pH) may be altered. Forinstance, the average surface charge and/or zeta potential of theparticles may be altered by changing the pH of a suspension containingthe particles to a pH that is greater than or equal to the pKa of one ormore components included in the particles (e.g., an ionizable molecule).In some such embodiments, the magnitude of the average surface chargeand/or zeta potential of the particles after changing the pH is greaterthan the magnitude prior to changing the pH. In some embodiments,altering one or more properties of the particles and/or suspension mayimprove yield, improve a characteristic of the resulting composition,and/or prevent or reduce certain problems, such as fouling during thefiltration process. For instance, the methods described herein mayresult in a composition including particles having less variations withrespect to one or more therapeutically-relevant parameters (e.g.,cross-sectional dimension, weight percentage of polynucleotide) comparedto a composition formed by certain existing methods.

A non-limiting process flow diagram of a method for preparing asuspension including particles comprising polynucleotides (e.g., mRNA)is shown illustratively in FIG. 1 . In some embodiments, a method 10 forpreparing a suspension may include forming steps, an altering step,and/or one or more filtration steps, amongst others. For instance, afirst solution 15 comprising polynucleotides (e.g., mRNA) and a secondsolution 20 comprising one or more components (e.g., ionizable molecule,sterol molecule) may be combined in step 22 to form a mixture 25comprising the polynucleotides and component(s). After the combiningstep, the mixture may undergo an incubation step 27. In someembodiments, the incubation step may comprise one or more dilutionsteps. In some instances, a dilution step may be used to alter theconcentration of one or more component (e.g., organic solvent) in themixture. Over time, the polynucleotides and component(s) mayspontaneously form, and/or be induced to form, a suspension containingparticles 30. For example, the composition of the first and/or secondsolutions (e.g., solvents) may be selected to induce co-precipitation ofthe polynucleotides (e.g., mRNA) and one or more components (e.g.,ionizable molecule) during an incubation step 27 to form particles 30.

In some embodiments, the incubation time, timing of dilution, ratio ofthe first and second solutions, and/or another condition (e.g.,concentration of polynucleotides, pH) may be controlled to influence oneor more properties of the resulting particles. For instance, theincubation time (i.e., the time allowed for the first and secondsolutions to mix or combine before a subsequent process step) may becontrolled to influence the weight percentage of polynucleotide (e.g.,mRNA) in the particles. In certain embodiments, the dilution timing maybe controlled to tailor the average cross-sectional dimension of theparticles. For instance, particle growth may be quenched after a certainperiod of time via a dilution. Methods for controlling the incubationtime and other features are described in more detail below.

As shown illustratively in FIG. 1 , the method may also involve alteringone or more properties of the particles in a step 32 to form alteredparticles 35, as described in more detail below. Subsequently, thealtered particles may be filtered during a filtering step 37 to form afiltered suspension containing particles 40.

Referring back to the step of forming the suspension containingparticles 30, the particles may be formed by any suitable method. Insome embodiments, the particle formation steps may be performed in afluidic device, such as a microfluidic device. For instance, a firstchannel may contain a first solution and a second channel may contain asecond solution. In some such embodiments, the combination step may beperformed using a device comprising a T junction that merges the firstand second channels, as described in U.S. Provisional Patent ApplicationSer. No. 62/040,989, filed Aug. 22, 2014, entitled “Lipid Nanoparticlesfor Nucleic Acid Molecules and Uses Thereof,” which is incorporated byreference in its entirety.

Other methods of forming the particles described herein are alsopossible. For instance, in embodiments in which the particles areliposomes, methods of forming liposomes can be used as described in moredetail below.

In some embodiments, the particles may be formed such that one or moreof the components (e.g., initially contained in the second solution) atleast partially (e.g., partially, completely) encapsulates at least aportion of the polynucleotides (e.g., initially contained in the firstsolution). For instance, in some embodiments, the particles comprise acomponent in the form of an ionizable molecule (e.g., an ionizablelipid). The ionizable molecules may encapsulate (e.g., partially,completely) at least a portion of the polynucleotides. In some cases,substantially all of the polynucleotides in the particles areencapsulated by the ionizable molecules. For example, the particles mayinclude an outer layer of the ionizable molecules with thepolynucleotides positioned in the interior of the particles. In otherembodiments, a portion of the polynucleotides may be exposed at thesurface of the particles, but another portion of the polynucleotides maybe encapsulated within the interior of the particles. In certainembodiments, the particles comprise two or more components (e.g., anionizable lipid, at least one other lipid, and/or at least one sterol)that together encapsulate (e.g., partially, completely) at least aportion of the polynucleotides. In some cases, substantially all of thepolynucleotides in the particles are encapsulated by the two or morecomponents. For instance, the polynucleotides may be encapsulated (e.g.,partially, completely) by an ionizable lipid, at least one other lipid,and/or at least one sterol. A particle comprising polynucleotides andone or more lipid molecules may be referred to as a lipid particle.

In some embodiments, particles 30 may be formed such that one or morecomponents of the particles may serve to at least partially (e.g.,partially, completely) shield and/or neutralize the charge of thepolynucleotides. In such cases, the magnitude of charge at the surfaceof the particles and/or the zeta potential of the particles may berelatively small. For instance, in embodiments in which the particlescomprise an ionizable molecule, at least a portion of the charge of thepolynucleotides may be shielded by the ionizable molecules. In some suchembodiments, the ionizable molecule may be oppositely charged withrespect to the overall charge of the polynucleotides.

In some embodiments, such particles having a relatively low surfacecharge may be difficult to filter (e.g., based on size) under certainconditions. For instance, passing the particles through a poroussubstrate to separate particles based on size and/or to remove certaincontaminants may result in fouling of the porous substrate. In some suchcases, fouling may limit the efficacy of separation, prevent theformation of a filtered suspension comprising particles that have arelatively narrow distribution in cross-sectional dimension, and/orallow a significant amount of certain contaminants to remain. In certainapplications, such a variance in the cross-sectional dimension and/orconcentration of certain contaminants may limit the utility of theparticles. In some instances, the filtered suspension may have toundergo further complex, time-consuming, and/or costly all of ypurification steps prior to utilization.

It has been discovered within the context of certain embodimentsdescribed herein that altering one or more properties of the particlesand/or suspension prior to and/or during filtration, as indicated bystep 32 in FIG. 1 , can reduce and/or eliminate certain problemsassociated with filtration, such as fouling. In some embodiments, thealtering step may increase the magnitude of the average surface chargeand/or zeta potential of the particles. This change can increase therepulsion forces between the particles during filtration. Increasing therepulsion forces can reduce or prevent the agglomeration of substances(e.g., particles, components, and/or polynucleotides) at, near, orwithin the pores of the porous substrate, thereby reducing the amount offouling of the porous substrate. In some instances, the altering stepmay allow efficacious sterile filtering of the suspension. In someinstances, the altering step may allow efficacious tangential flowfiltration, including ultrafiltration and diafiltration.

Additionally or alternatively, in some embodiments, altering one or moreproperties of the particles and/or suspension prior to and/or duringfiltration may allow the formation of particles 40 having a desirableproperty. For example, the particles may have a relatively smallcoefficient of variation (e.g., less than or equal to about 20%) in across-section dimension, a relatively high weight percentage ofencapsulated polynucleotides (e.g., greater than or equal to about 85%of encapsulated/bound polynucleotide vs. free polynucleotide), amongstother features, as described in more detail herein.

It should be appreciated that the steps shown in FIG. 1 can vary. Forexample, although FIG. 1 shows the altering step 32 as being performedbefore filtration step 37, in some embodiments the altering step mayoccur during the filtration step as described herein. In otherembodiments, the filtered suspension containing particles 40 may besubjected to one or more further processing steps 39 (e.g., alteringtonicity, altering pH, altering stabilizer concentration, altering ionicstrength) to obtain a suspension containing particles 45.

As described herein, a method for preparing a suspension includingparticles comprising polynucleotides (e.g., mRNA) may involve analtering step. In some embodiments in which the particles include anionizable molecule, the altering step may involve changing a pH of thesuspension from a first pH to a second pH that is greater than a pKa ofthe ionizable molecule. For instance, the pH of the suspension prior toan altering step (and/or during or after formation of the particles) maybe less than a pKa of the ionizable molecule. The pH of the suspensionmay then be altered by adding a suitable base to increase the pH of thesuspension to be greater than a pKa of the ionizable molecule.

In some embodiments, the pH of the suspension prior to an altering step(and/or during or after formation of the particles) may be less than orequal to about 6.5, less than or equal to about 6, less than or equal toabout 5.8, less than or equal to about 5.5, less than or equal to about5.2, less than or equal to about 5, less than or equal to about 4.8, orless than or equal to about 4.5. In some embodiments, the pH prior tothe altering step (and/or during or after formation of the particles)may be greater than or equal to about 3, greater than or equal to about3.2, greater than or equal to about 3.5, greater than or equal to about3.8, greater than or equal to about 4.0, greater than or equal to about4.2, greater than or equal to about 4.5, or greater than or equal toabout 5.0. Combinations of the above referenced ranges are also possible(e.g., greater than or equal to about 3 and less than or equal to about6). Other ranges are also possible. In some embodiments in which the pHis greater than the pKa prior to the altering step and/or duringparticle formation, one or more properties (e.g., weight percentage ofpolynucleotide, cross-sectional dimension, coefficient of variation incross-sectional dimension) of the particle may be adversely affected ormay make particle formation infeasible.

In some embodiments, the pH of the suspension after the altering step(e.g., a pH altering step) may be may be greater than or equal to about6, greater than or equal to about 6.2, greater than or equal to about6.4, greater than or equal to about 6.5, greater than or equal to about6.6, greater than or equal to about 6.8, greater than or equal to about7, greater than or equal to about 7.2, greater than or equal to about7.4, greater than or equal to about 7.4, greater than or equal to about7.5, greater than or equal to about 7.6, greater than or equal to about7.8, or greater than or equal to about 8. In some embodiments, the pHafter the altering step may be less than or equal to about 9.0, lessthan or equal to about 8.8, greater than or equal to about 8.6, lessthan or equal to about 8.5, greater than or equal to about 8.4, lessthan or equal to about 8.2, less than or equal to about 8.0, less thanor equal to about 7.8, greater than or equal to about 7.6, or less thanor equal to about 7.5. Combinations of the above referenced ranges arealso possible (e.g., greater than or equal to 6.5 and less than or equalto about 8, greater than or equal to 7 and less than or equal to about8). Other ranges are also possible.

In some embodiments, the pH of the suspension after the altering step(e.g., a pH altering step) may be within a certain range or may have acertain value greater than the pKa of one or more components (e.g.,ionizable molecule) in the particles. In certain embodiments, the pH ofthe suspension after the altering step may be greater than or equal toabout 0.2 pH units, greater than or equal to about 0.4 pH units, greaterthan or equal to about 0.5 pH units, greater than or equal to about 0.6pH units, greater than or equal to about 0.8 pH units, greater than orequal to about 1 pH unit, greater than or equal to about 1.2 pH units,greater than or equal to about 1.4 pH units, or greater than or equal toabout 1.5 pH units greater than the pKa of one or more components (e.g.,ionizable molecule) in the particles. In some instances, the pH of thesuspension after the altering step may be less than or equal to about2.0 pH units, less than or equal to about 1.8 pH units, less than orequal to about 1.5 pH units, less than or equal to about 1.2 pH units,less than or equal to about 1.0 pH unit, or less than or equal to about0.8 pH units greater than the pKa of one or more components (e.g.,ionizable molecule) in the particles. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 0.5pH units and less than or equal to about 2 pH units, greater than orequal to 1 pH unit and less than or equal to about 1.5 pH units). Otherranges are also possible. In some embodiments, the ionizable molecule isa cationic lipid such as2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319). Otherionizable molecules are described herein.

In certain embodiments, the altering step may involve changing anaverage zeta potential of the particles from a first zeta potential to asecond zeta potential that is greater than the magnitude of the firstzeta potential.

In some embodiments, the average zeta potential of the particles priorto an altering step (and/or during or after formation of the particles)may be positive. In some such embodiments, the average zeta potential ofthe particles prior to an altering step (and/or during or afterformation of the particles) may be greater than or equal to about +0.1mV, greater than or equal to about +0.5 mV, greater than or equal toabout +1.0 mV, greater than or equal to about +1.5 mV, greater than orequal to about +2.0 mV, greater than or equal to about +5.0 mV, greaterthan or equal to about +10.0 mV, greater than or equal to about +15.0mV, greater than or equal to about +20.0 mV, or greater than or equal toabout +25 mV. In some such cases, the average zeta potential of theparticles prior to an altering step (and/or during or after formation ofthe particles) may be less than or equal to about +30 mV, less than orequal to about +25 mV, less than or equal to about +20 mV, less than orequal to about +15 mV, less than or equal to about +10 mV, less than orequal to about +5 mV, less than or equal to about +3 mV, or 0 mV.Combinations of the above-referenced ranges are also possible (e.g.,greater than or equal to about +1.0 mV and less than or equal to about+30 mV).

In other embodiments, the average zeta potential of the particles priorto an altering step (and/or during or after formation of the particles)may be negative. In some such embodiments, the average zeta potential ofthe particles may be greater than or equal to about −2 mV, greater thanor equal to about −1.8 mV, greater than or equal to about −1.5 mV,greater than or equal to about −1.2 m, greater than or equal to about −1mV, greater than or equal to about −0.8 mV, or greater than or equal toabout −0.5 mV. In some cases, the average zeta potential of theparticles prior to an altering step (and/or during or after formation ofthe particles) may be 0 mV or less than or equal to 0.1 mV. Combinationsof the above-referenced ranges are also possible (e.g., greater than orequal to about −2 mV and less than or equal to 0.1 mV).

In some embodiments, the average zeta potential of the particles afteran altering step may be less than or equal to about −2 mV, less than orequal to about −3 mV, less than or equal to about −5 mV, less than orequal to about −8 mV, less than or equal to about −10 mV, less than orequal to about −12 mV, less than or equal to about −15 mV, greater thanor equal to about −18 mV, less than or equal to about −20 mV, less thanor equal to about −22 mV, less than or equal to about −25 mV, less thanor equal to about −28 mV, less than or equal to about −30 mV, or lessthan or equal to about −35 mV. The average zeta potential of theparticles after an altering step may be greater than or equal to about−40 mV, greater than or equal to about −38 mV, greater than or equal toabout −35 mV, greater than or equal to about −30 mV, greater than orequal to about −28 mV, greater than or equal to about −25 mV, greaterthan or equal to about −22 mV, greater than or equal to about −20 mV,greater than or equal to about −18 mV, greater than or equal to about−15 mV, greater than or equal to about −12 mV, greater than or equal toabout −10 mV, greater than or equal to about −8 mV. Combinations of theabove referenced ranges are also possible (e.g., less than or equal to−1 mV and greater than or equal to about −10 mV). Other ranges are alsopossible.

In some embodiments, the average zeta potential of the particles may benegative after the altering step. In some such embodiments, the averagezeta potential of the particles comprising an ionizable molecule and atleast one polynucleotides may be less than the average zeta potential ofessentially identical particle that do not comprise a polynucleotide. Itshould be understood that as used herein, when the zeta potential ofparticles are compared (e.g., less than, greater than), the comparisonis based on the value of the zeta potential and not the magnitude. Forinstance, a zeta potential of −30 mV is less than a zeta potential of −1mV.

In some instances, a change in pH of a suspension may cause a change inthe magnitude and/or polarity of the surface charge, and accordingly thezeta potential, of the particles, as illustrated in FIG. 2 . FIG. 2illustratively shows the surface charge of particles in a suspension ata pH below the pKa of one or more components of the particles (e.g., anionizable molecule) and at a pH above the pKa of one or more componentsof the particles. As illustrated in FIG. 2 , the sign of the surfacecharge of the particles at a pH below the pKa may be opposite of thesign of the surface charge of the particles at a pH above the pKa. Itshould be understood that FIG. 2 is illustrative and that the sign ofthe surface charge of the particles need not be at a pH below the pKaneed not be opposite of the sign of the surface charge of the particlesat a pH above the pKa in all embodiments. In some embodiments, theaverage magnitude of the surface charge of the particles before thealtering step may be greater than the magnitude the average magnitude ofthe surface charge of the particles after the altering step. In somesuch embodiments, the average magnitude of surface charge of theparticles after the altering step may be greater than before thealtering step. In certain embodiments, regardless of the averagemagnitude and/or polarity of the surface charge after the altering step,the altering step may serve to increase the repulsive force betweenparticles.

Without being bound by theory, it is believed that the change in surfacecharge of the particles as a function of pH may be due, at least inpart, to deprotonation of the ionizable lipid. For example, when theionizable lipid is positively charged, at pH values less than the pKa,the positively charged ionizable lipid components bind negativelycharged species on the surface and present an overall stoichiometricexcess of positive charge. As the positive charge is diminished at pHvalues greater than the pKa, surface-accessible negatively chargedgroups (e.g., lipids, nucleotides) are no longer neutralized and candominate the charge profile.

In some embodiments, and as shown illustratively in FIG. 3 , afterformation of a particle 50 and/or prior to an altering step, particle 50may be arranged such that ionizable molecules 55 at least partiallyencapsulate polynucleotides 60. The ionizable molecule may comprise acharged portion (e.g., a nitrogen-containing functional group) thatshields the charge of the polynucleotide, resulting in a relativelysmall charge at a surface 67 of the particle. Particle 50 may be presentin a suspension, in some embodiments, prior to altering the pH of thesuspension to a pH that is greater than the pKa of the ionizablemolecule. In some embodiments, altering the pH of the suspension to a pHthat is greater than the pKa of the ionizable molecule may alter thecharge state of the charged portion of the ionizable molecule. Forexample, the percentage of charged ionizable molecules (e.g., ionizablelipids) may be reduced for positively charged ionizable molecules. Insome embodiments, the change in the charge state of the charged portionof the ionizable molecule may result in reorganization of the particlecomponents to form an altered particle 70. For example, as shown in FIG.3 , reorganization may result in at least a portion of thepolynucleotides being present on the surface of particle 70. Thepresence of polynucleotides on the surface of the particle may increasethe magnitude of the surface charge. For instance, the polynucleotideson the surface may make the particles have a greater surface charge. Inother embodiments, the change in the charge state of the charged portionof the ionizable molecule may not result in structural reorganization ofthe particle. In some such embodiments, as the pH is adjusted and thecharge state of ionizable molecule is diminished, the balance shiftsresulting in a change in sign for zeta potential. In some such cases,the change in the ionization of charged groups on the surface of theparticles results in a change in zeta potential by a shift in counterion solvation around the molecules in the particle.

It should be understood that FIG. 3 is non-limiting and other particleconfigurations are possible. For instance, in some embodiments theionizable molecules in particle 50 may have a different configurationcompared to their configuration in particle 70. In some cases, particles50 and 70 are liposomes (i.e., liposomal particles). In some such cases,the particles may comprise a lipid bilayer surrounding an aqueousinterior. In some embodiments, liposomes can be, but not limited to, amultilamellar vesicle (MLV) and may contain a series of concentricbilayers separated by narrow aqueous compartments or an unicellularvesicle.

In some cases, particles including certain components may be more proneto fouling during filtration compared to particles including othercomponents. In certain embodiments, particles prone to fouling may havea relatively low or neutral surface charge (e.g., particle 50 of FIG. 3). Causing the particles to rearrange to have an overall greater surfacecharge (e.g., particle 70 of FIG. 3 ) may reduce fouling as described inmore detail herein.

In some embodiments, a method described herein comprises certainparticle formation steps. In some such embodiments, the formation stepsmay comprise a combination step and/or an incubation step. Thecombination step may comprise mixing an aqueous solution comprising abuffer and polynucleotides (e.g., mRNA) (e.g., a first solution) with asolution comprising a solvent such as an organic solvent (e.g., analcohol) and particle components (e.g., ionizable molecules, sterol)(e.g., a second solution). After the combination step, the suspensionmay be incubated to allow for particle formation. In some embodiments,one or more properties of the particles and/or resulting composition,such as weight percentage of polynucleotides (e.g., mRNA) in theparticle, may be improved by tuning the incubation time. The incubationtime may be measured from the time of combining the first and secondsolutions until the time of a subsequent process step (e.g., analteration step). In some embodiments, the subsequent process step isaltering the pH of the suspension to a pH greater than the pKa of acomponent (e.g., ionizable molecule) of the particles, such that theincubation time is measured from the time of combining the first andsecond solutions until the pH of the suspension is greater than the pKaa component of the particles.

An incubation time as described herein may vary. For instance, in someembodiments, the incubation time may be greater than or equal to about 3minutes, greater than or equal to about 4 minutes, greater than or equalto about 5 minutes, greater than or equal to about 6 minutes, greaterthan or equal to about 8 minutes, greater than or equal to about 10minutes, greater than or equal to about 12 minutes, or greater than orequal to about 15 minutes. In some embodiments, the incubation time maybe less than or equal to about 30 minutes, less than or equal to about20 minutes, less than or equal to about 18 minutes, less than or equalto about 15 minutes, less than or equal to about 12 minutes, less thanor equal to about 10 minutes, less than or equal to about 8 minutes, orless than or equal to about 6 minutes. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 5minutes and less than or equal to about 20 minutes). Other ranges arealso possible.

In some embodiments, the incubation step may comprise one or moredilution steps (e.g., two dilution steps). In some embodiments, adilution step may be used to alter the concentration of one or morecomponents in the mixture (e.g., organic solvent). In some embodiments,the dilution step may facilitate particle formation. In certainembodiments, one or more dilution steps may adjust the pH of themixture. In such embodiments, the pH of the mixture after the dilutionstep may be altered to be less than the pKa of one or more components(e.g., ionizable molecules) in the mixture.

In some embodiments, a dilution step may be used to slow down, limit,and/or quench particle growth. That is, in some embodiments, a dilutionstep may be used to control the average cross-sectional dimension of theparticles. In certain embodiments, the incubation step may comprise twodilution steps. For instance, one dilution step may be used to changethe concentration of a component in the mixture and another dilutionstep may be used to control (e.g., quench) particle growth.

As described herein, in some cases, an altering step (e.g., altering pH)may be performed. The altering step may be performed at any suitabletime after one or more of the particle formation steps (e.g., aformation step, a combining step, an incubation step). For instance, insome embodiments, the time of an altering step may be greater than orequal to about 1 minute, greater than or equal to about 3 minutes,greater than or equal to about 5 minutes, greater than or equal to about8 minutes, greater than or equal to about 10 minutes, greater than orequal to about 12 minutes, greater than or equal to about 15 minutes,greater than or equal to about 18 minutes, greater than or equal toabout 20 minutes, or greater than or equal to about 25 minutes after oneor more of the particle forming steps (e.g., a formation step, acombining step, an incubation step). In some embodiments, an alteringstep may be performed less than or equal to about 30 minute, less thanor equal to about 28 minutes, less than or equal to about 25 minutes,less than or equal to about 22 minutes, less than or equal to about 20minute, less than or equal to about 18 minutes, less than or equal toabout 15 minutes, less than or equal to about 12 minutes, less than orequal to about 10 minutes, less than or equal to about 8 minutes, orless than or equal to about 6 minutes after one or more of the particleforming steps (e.g., a formation step, a combining step, an incubationstep). Combinations of the above referenced ranges are also possiblee.g., greater than or equal to 5 minutes and less than or equal to about30 minutes).

In some embodiments, a method described herein may allow the particlesto have a relatively high encapsulation efficiency of a component of theparticle (e.g., a polynucleotide, sterol). For instance, in someembodiments, the encapsulation efficiency of a component may be greaterthan or equal to about 50%, greater than or equal to about 55%, greaterthan or equal to about 60%, greater than or equal to about 65%, greaterthan or equal to about 70%, greater than or equal to about 75%, greaterthan or equal to about 80%, greater than or equal to about 85%, greaterthan or equal to about 90%, greater than or equal to about 95%, orgreater than or equal to about 99%. In some instances, the encapsulationefficiency of a component may be less than or equal to about 100%, lessthan or equal to about 99%, less than or equal to about 95%, less thanor equal to about 90%, less than or equal to about 85%, less than orequal to about 80%, less than or equal to about 75%, less than or equalto about 70%, less than or equal to about 65%, or less than or equal toabout 60%. Combinations of the above referenced ranges are also possible(e.g., greater than or equal to 75% and less than or equal to about100%). The encapsulation efficiency is determined by the percentage(e.g., by weight, mol) of the component in the particles compared to theinitial amount of the component used prior to particle formation andpurification. In embodiments in which more than one component isincluded in the particles, each component may independently have anencapsulation efficiency in one or more of the above-referenced ranges.

As noted above, a method described herein may comprise one or morefiltration steps. In some embodiments, the one or more filtration stepsmay include tangential flow filtration (e.g., cross-flow filtration witha membrane), clarifying filtration, and/or sterile filtration (e.g.,with a 0.2 micron filter). In some embodiments, the methods stepsdescribed herein may prevent or reduce fouling during tangential flowfiltration and/or sterile filtration. Other filtration methods are alsopossible, as described in more detail herein.

In some embodiments, the filtration step (e.g., tangential flowfiltration) may include one or more concentration filtration steps andone or more diafiltration (e.g., buffer exchange, wash) steps. Forinstance, the filtration step may include two or more concentrationfiltration steps and a diafiltration step. In some such cases, aconcentration step may be performed before and after the diafiltrationstep. As used herein, the term “concentration filtration step” has itsordinary meaning in the art and refers to a filtration step in which theconcentration of one or more components in the collected fraction isgreater than the concentration of component(s) in the original feed. Insome embodiments, the collected fraction may be the permeate, alsoreferred to as the filtrate. In some embodiments, the collected fractionis the retentate, also referred to as the concentrate. In someembodiments, the concentration filtration may be ultrafiltration. Asused herein, the term “diafiltration” has its ordinary meaning in theart and refers to a technique that uses a filter to remove, replace, orlower the concentration of one or more components in the feed (e.g.,salts, solvent). In some embodiments, a filtration step may include adiafiltration step that involves passing a certain volume (e.g.,diafiltration volume) of a fluid through the filter and/or filtrationsystem. As used herein, the term “diafiltration volume” or “diavolume”has its ordinary meaning in the art and may refer to the volume ofretentate (e.g., total volume of reservoir plus the hold-up volume oftubing, filter, holder, etc.) at the start of diafiltration.

In some embodiments, multiple diafiltration volumes may be passedthrough the filter and/or filtration system during the filtration step.For instance, in some embodiments, greater than or equal to about onediafiltration volume, greater than or equal to about two diafiltrationvolumes, greater than or equal to about three diafiltration volumes,greater than or equal to about four diafiltration volumes, greater thanor equal to about five diafiltration volumes, greater than or equal toabout six diafiltration volumes, greater than or equal to about eightdiafiltration volumes, greater than or equal to about ten diafiltrationvolumes, greater than or equal to about twelve diafiltration volumes, orgreater than or equal to about fifteen diafiltration volumes may be usedduring a diafiltration step. In some instances, less than or equal toabout twenty diafiltration volumes, less than or equal to about tendiafiltration volumes, less than or equal to about eight diafiltrationvolumes, less than or equal to about seven diafiltration volumes, lessthan or equal to about six diafiltration volumes, less than or equal toabout five diafiltration volumes, less than or equal to about fourdiafiltration volumes, or less than or equal to about threediafiltration volumes may be used during a diafiltration step.Combinations of the above-referenced ranges are also possible (e.g.,greater than or equal to about two diafiltration volumes and less thanor equal to about ten diafiltration volumes, greater than or equal toabout five diafiltration volumes and less than or equal to about eightdiafiltration volumes).

As described herein, the disclosed method(s) may result in a reductionof fouling during filtration. In some embodiments, the reduction infouling during filtration may allow a relatively high permeate flux tobe achieved and/or may allow a relatively high permeate flux to bemaintained throughout the filtration step or at least a portion of thefiltration step (e.g., during a concentration step and/or diafiltrationstep). As used herein, the term “permeate flux” has its ordinary meaningin the art and refers to the rate of sample flow through a given filterarea per unit time.

In some embodiments, the permeate flux during the filtration step and/orat least a portion of the filtration step (e.g., during concentrationstep(s) and/or a diafiltration step, e.g., after at least about 5diafiltration volumes) at a transmembrane pressure of between about 1psi and about 20 psi (e.g., between about 10 psi and about 15 psi) maybe greater than or equal to about 10 L/m²hr, greater than or equal toabout 20 L/m²hr, greater than or equal to about 30 L/m²hr, greater thanor equal to about 40 L/m²hr, greater than or equal to about 50 L/m²hr,greater than or equal to about 60 L/m²hr, greater than or equal to about70 L/m²hr, greater than or equal to about 80 L/m²hr, greater than orequal to about 90 L/m²hr, greater than or equal to about 100 L/m²hr, orgreater than or equal to about 110 L/m²hr. In some instances, thepermeate flux during the filtration step and/or at least a portion ofthe filtration step (e.g., concentration step(s) and/or diafiltrationstep, e.g., after 5 diafiltration volumes) at a transmembrane pressureof between about 1 psi and about 20 psi (e.g., between about 10 psi andabout 15 psi) may be less than or equal to about 120 L/m²hr, less thanor equal to about 110 L/m²hr, less than or equal to about 100 L/m²hr,less than or equal to about 90 L/m²hr, less than or equal to about 80L/m²hr, less than or equal to about 70 L/m²hr, less than or equal toabout 60 L/m²hr, less than or equal to about 50 L/m²hr, less than orequal to about 40 L/m²hr, less than or equal to about 30 L/m²hr, or lessthan or equal to about 20 L/m²hr. Combinations of the above-referencedranges are also possible (e.g., greater than or equal to about 10 L/m²hrand less than or equal to about 120 L/m²hr, greater than or equal toabout 20 L/m²hr and less than or equal to about 110 L/m²hr).

In some embodiments, the particles comprise one or more of ionizablemolecules, polynucleotides, and optional components, such as sterols,neutral lipids, and a molecule capable of reducing particle aggregation(e.g., polyethylene glycol (PEG), polyethylene glycol-modified lipid).

In some embodiments, a particle described herein may include one or moreionizable molecules. The ionizable molecule may comprise a charged groupand may have a certain pKa. In certain embodiments, the pKa of theionizable molecule may be greater than or equal to about 6, greater thanor equal to about 6.2, greater than or equal to about 6.5, greater thanor equal to about 6.8, greater than or equal to about 7, greater than orequal to about 7.2, greater than or equal to about 7.5, greater than orequal to about 7.8, greater than or equal to about 8. In someembodiments, the pKa of the ionizable molecule may be less than or equalto about 10, less than or equal to about 9.8, less than or equal toabout 9.5, less than or equal to about 9.2, less than or equal to about9.0, less than or equal to about 8.8, or less than or equal to about8.5. Combinations of the above referenced ranges are also possible(e.g., greater than or equal to 6 and less than or equal to about 8.5).Other ranges are also possible. In embodiments in which more than onetype of ionizable molecules are present in a particle, each type ofionizable molecule may independently have a pKa in one or more of theranges described above.

In general, an ionizable molecule comprises one or more charged groups.In some embodiments, an ionizable molecule may be positively charged ornegatively charged. For instance, an ionizable molecule may bepositively charged. For example, an ionizable molecule may comprise anamine group. As used herein, the term “ionizable molecule” has itsordinary meaning in the art and may refer to a molecule or matrixcomprising one or more charged moiety. As used herein, a “chargedmoiety” is a chemical moiety that carries a formal electronic charge,e.g., monovalent (+1, or −1), divalent (+2, or −2), trivalent (+3, or−3), etc. The charged moiety may be anionic (i.e., negatively charged)or cationic (i.e., positively charged). Examples of positively-chargedmoieties include amine groups (e.g., primary, secondary, and/or tertiaryamines), ammonium groups, pyridinium group, guanidine groups, andimidizolium groups. In a particular embodiment, the charged moietiescomprise amine groups. Examples of negatively-charged groups orprecursors thereof, include carboxylate groups, sulfonate groups,sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups,and the like. The charge of the charged moiety may vary, in some cases,with the environmental conditions, for example, changes in pH may alterthe charge of the moiety, and/or cause the moiety to become charged oruncharged. In general, the charge density of the molecule and/or matrixmay be selected as desired.

In some cases, an ionizable molecule may include one or more precursormoieties that can be converted to charged moieties. For instance, theionizable molecule may include a neutral moiety that can be hydrolyzedto form a charged moiety, such as those described above. As anon-limiting specific example, the molecule or matrix may include anamide, which can be hydrolyzed to form an amine, respectively. Those ofordinary skill in the art will be able to determine whether a givenchemical moiety carries a formal electronic charge (for example, byinspection, pH titration, ionic conductivity measurements, etc.), and/orwhether a given chemical moiety can be reacted (e.g., hydrolyzed) toform a chemical moiety that carries a formal electronic charge.

It should be understood that the terms “charged” or “charged moiety”does not refer to a “partial negative charge” or “partial positivecharge” on a molecule. The terms “partial negative charge” and “partialpositive charge” are given its ordinary meaning in the art. A “partialnegative charge” may result when a functional group comprises a bondthat becomes polarized such that electron density is pulled toward oneatom of the bond, creating a partial negative charge on the atom. Thoseof ordinary skill in the art will, in general, recognize bonds that canbecome polarized in this way.

In some embodiments, an ionizable molecule is a lipid. For instance, theionizable molecule may be a natural or synthetic lipid or lipid analog(i.e., lipophilic molecule). Non-limiting examples of natural orsynthetic lipids or lipid analogs include fatty acyls, glycerolipids,glycerophospholipids, sphingolipids, saccharolipids and polyketides(derived from condensation of ketoacyl subunits), and sterol lipids andprenol lipids (derived from condensation of isoprene subunits). Otherionizable molecules are also possible.

In some embodiments, the ionizable molecule is a charged lipid. Forinstance, in some embodiments, the ionizable molecule is a cationiclipid. In one embodiment, the cationic lipid may have a positivelycharged hydrophilic head and a hydrophobic tail that are connected via alinker structure. Non-limiting examples of cationic lipids includeC12-200, DLin-DMA (1,2-dilinoleyloxy-3-dimethylaminopropane),DLin-K-DMA, DODMA (1,2-dioleyloxy-N,N-dimethylaminopropane),DLin-MC3-DMA, DLin-KC2-DMA,2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]−2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US20130150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-10-yloxy]methyl}propan-1-ol(Compound 2 in US20130150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US20130150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US20130150625),(20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine,(1Z,19Z)-N5N-dimethylpentacosa-16, 19-dien-8-amine,(13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)-N,N-dimetylheptacos-18-en-10-amine,(17Z)-N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)-N,N-dimethylheptacos-20-en-10-amine,(15Z)-N,N-dimethyleptacos-15-en-10-amine,(14Z)-N,N-dimethylnonacos-14-en-10-amine,(17Z)-N,N-dimethylnonacos-17-en-10-amine,(24Z)-N,N-dimethyltritriacont-24-en-10-amine,(20Z)-N,N-dimethylnonacos-20-en-10-amine,(22Z)-N,N-dimethylhentriacont-22-en-10-amine,(16Z)-N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,(13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-hepty lcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-imethylpentadecan-6-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)-N,N-dimethyl-H(1-metoyloctyl)oxyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amineand (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine, andpharmaceutically acceptable salts or stereoisomers thereof. Inembodiments in which more than one type of ionizable molecules arepresent in a particle, each type of ionizable molecule may independentlybe chosen from the ionizable molecules described herein.

In some embodiments, an ionizable cationic lipid is selected from thegroup consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), andpharmaceutically acceptable salts or stereoisomers thereof.

In some embodiments, the ionizable molecule comprises a nitrogen atom.In some such embodiments, a molar ratio of nitrogen atoms in theionizable molecules to the phosphates in the polynucleotides (N:P ratio)may be greater than or equal to about 1:1, greater than or equal toabout 2:1, greater than or equal to about 3:1, greater than or equal toabout 5:1, greater than or equal to about 8:1, greater than or equal toabout 10:1, greater than or equal to about 12:1, greater than or equalto about 15:1, or greater than or equal to about 20:1. In someembodiments, the N:P ratio may be less than or equal to about 20:1, lessthan or equal to about 18:1, less than or equal to about 15:1, less thanor equal to about 12:1, less than or equal to about 10:1, less than orequal to about 8:1, or less than or equal to about 5:1 Combinations ofthe above referenced ranges are also possible (e.g., greater than orequal to 1:1 and less than or equal to about 20:1). Other ranges arealso possible. In embodiments in which more than one type of ionizablemolecules are present in a particle, each type of ionizable molecule mayindependently have a N:P ratio in one or more of the ranges describedabove.

In some embodiments, the ionizable molecule may be a lipid bound to apolycation. Non-limiting examples of polycations may include naturalpolycations (e.g., chitosan), synthetic polycations (e.g., polyamines,such as polyethylene imine) a cationic peptide or a polypeptide such as,but not limited to, polylysine, polyornithine and/or polyarginine.

The ionizable molecule may have any suitable molecular weight. Incertain embodiments, the molecular weight of an ionizable molecule isless than or equal to about 2,500 g/mol, less than or equal to about2,000 g/mol, less than or equal to about 1,500 g/mol, less than or equalto about 1,250 g/mol, less than or equal to about 1,000 g/mol, less thanor equal to about 900 g/mol, less than or equal to about 800 g/mol, lessthan or equal to about 700 g/mol, less than or equal to about 600 g/mol,less than or equal to about 500 g/mol, less than or equal to about 400g/mol, less than or equal to about 300 g/mol, less than or equal toabout 200 g/mol, or less than or equal to about 100 g/mol. In someinstances, the molecular weight of an ionizable molecule is greater thanor equal to about 100 g/mol, greater than or equal to about 200 g/mol,greater than or equal to about 300 g/mol, greater than or equal to about400 g/mol, greater than or equal to about 500 g/mol, greater than orequal to about 600 g/mol, greater than or equal to about 700 g/mol,greater than or equal to about 1000 g/mol, greater than or equal toabout 1,250 g/mol, greater than or equal to about 1,500 g/mol, greaterthan or equal to about 1,750 g/mol, greater than or equal to about 2,000g/mol, or greater than or equal to about 2,250 g/mol. Combinations ofthe above ranges (e.g., at least about 200 g/mol and less than or equalto about 2,500 g/mol) are also possible. In embodiments in which morethan one type of ionizable molecules are present in a particle, eachtype of ionizable molecule may independently have a molecular weight inone or more of the ranges described above.

In some embodiments, the percentage (e.g., by weight, or by mole) of asingle type of ionizable molecule and/or of all the ionizable moleculeswithin a particle may be greater than or equal to about 30%, greaterthan or equal to about 35%, greater than or equal to about 40%, greaterthan or equal to about 42%, greater than or equal to about 45%, greaterthan or equal to about 48%, greater than or equal to about 50%, greaterthan or equal to about 52%, greater than or equal to about 55%, greaterthan or equal to about 58%, greater than or equal to about 60%, greaterthan or equal to about 62%, greater than or equal to about 65%, orgreater than or equal to about 68%. In some instances, the percentage(e.g., by weight, or by mole) may be less than or equal to about 70%,less than or equal to about 68%, less than or equal to about 65%, lessthan or equal to about 62%, less than or equal to about 60%, less thanor equal to about 58%, less than or equal to about 55%, less than orequal to about 52%, less than or equal to about 50%, or less than orequal to about 48%. Combinations of the above referenced ranges are alsopossible (e.g., greater than or equal to 30% and less than or equal toabout 70%, greater than or equal to 40% and less than or equal to about65%). In embodiments in which more than one type of ionizable moleculesare present in a particle, each type of ionizable molecule mayindependently have a percentage (e.g., by weight, or by mole) in one ormore of the ranges described above. The percentage (e.g., by weight, orby mole) may be determined by extracting the ionizable molecule(s) fromthe dried particles using, e.g., organic solvents, and measuring thequantity of the agent using high pressure liquid chromatography (i.e.,HPLC), liquid chromatography-mass spectrometry, nuclear magneticresonance, or mass spectrometry. Those of ordinary skill in the artwould be knowledgeable of techniques to determine the quantity of acomponent using the above-referenced techniques. For example, HPLC maybe used to quantify the amount of a component, by, e.g., comparing thearea under the curve of a HPLC chromatogram to a standard curve.

As described herein, a particle may include a polynucleotide, i.e., apolymer of nucleotides. In some embodiments, a particle includes morethan one (e.g., at least 2, 3, 4, 5, 6, etc. types of polynucleotides).Typically, a polynucleotide comprises at least three nucleotides. Thepolymer may include natural nucleosides (i.e., adenosine, thymidine,guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine,deoxyguanosine, and deoxycytidine), nucleoside analogs (e.g.,2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, C5-propynylcytidine, C5-propynyluridine, C5 bromouridine, C5fluorouridine, C5 iodouridine, C5 5 methylcytidine, 7 deazaadenosine, 7deazaguanosine, 8 oxoadenosine, 8 oxoguanosine, O(6) methylguanine, and2-thiocytidine), chemically modified bases, biologically modified bases(e.g., methylated bases), intercalated bases, modified sugars (e.g.,2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose), ormodified phosphate groups (e.g., phosphorothioates and 5′ Nphosphoramidite linkages). In some embodiments, the polynucleotide ismRNA, or alternative or modified mRNA. In some embodiments, thepolynucleotide comprises at least about 50 nucleotides, at least about100 nucleotides, at least about 250 nucleotides, at least about 500nucleotides, at least about 750 nucleotides, at least about 1,000nucleotides, at least about 1,500 nucleotides, at least about 2,000nucleotides, at least about 2,500 nucleotides, at least about 3,000nucleotides, at least about 3,500 nucleotides or at least about 4,000nucleotides and less than about 10,000 nucleotides (e.g., between about100 and about 5,000 nucleotides).

In some embodiments, the particle comprises one or more sterols (e.g.,cholesterol). A non-limiting example of a sterol include cholesterol.

The sterol molecules may have any suitable molecular weight. In certainembodiments, the molecular weight of a sterol molecule may be less thanor equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol,less than or equal to about 1,500 g/mol, less than or equal to about1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equalto about 900 g/mol, less than or equal to about 800 g/mol, less than orequal to about 700 g/mol, less than or equal to about 600 g/mol, lessthan or equal to about 500 g/mol, less than or equal to about 400 g/mol,less than or equal to about 300 g/mol, less than or equal to about 200g/mol, or less than or equal to about 100 g/mol. In some instances, themolecular weight of a sterol molecule may be greater than or equal toabout 100 g/mol, greater than or equal to about 200 g/mol, greater thanor equal to about 300 g/mol, greater than or equal to about 400 g/mol,greater than or equal to about 500 g/mol, greater than or equal to about600 g/mol, greater than or equal to about 700 g/mol, greater than orequal to about 1000 g/mol, greater than or equal to about 1,250 g/mol,greater than or equal to about 1,500 g/mol, greater than or equal toabout 1,750 g/mol, greater than or equal to about 2,000 g/mol, orgreater than or equal to about 2,250 g/mol. Combinations of the aboveranges (e.g., at least about 200 g/mol and less than or equal to about2,500 g/mol) are also possible. In embodiments in which more than onetype of sterol are present in a particle, each type of sterol mayindependently have a molecular weight in one or more of the rangesdescribed above.

In some embodiments, the percentage (e.g., by weight, or by mole) of asingle type of sterol (e.g., cholesterol) and/or of all the sterols in aparticle may be greater than or equal to about 0.5%, greater than orequal to about 1%, greater than or equal to about 2%, greater than orequal to about 4%, greater than or equal to about 6%, greater than orequal to about 8%, greater than or equal to about 10%, greater than orequal to about 15%, greater than or equal to about 20%, greater than orequal to about 25%, greater than or equal to about 30%, greater than orequal to about 35%, greater than or equal to about 40%, greater than orequal to about 45%, or greater than or equal to about 50%. In someinstances, the percentage (e.g., by weight, or by mole) may be less thanor equal to about 60%, less than or equal to about 55%, less than orequal to about 50%, less than or equal to about 45%, less than or equalto about 40%, less than or equal to about 35%, less than or equal toabout 30%, less than or equal to about 25%, less than or equal to about20%, or less than or equal to about 15%. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 30%and less than or equal to about 60%). In embodiments in which more thanone type of sterol are present in a particle, each type of sterol mayindependently have a percentage (e.g., by weight, or by mole) withrespect to the particle in one or more of the ranges described above.The percentage (e.g., by weight, or by mole) may be determined asdescribed above with respect to ionizable molecules.

In some embodiments, the particle comprises one or more neutral lipids(i.e., neutrally-charged lipids). Non-limiting examples of neutrallipids include DSPC, DPPC, POPC, DOPE and SM.

In some embodiments, a lipid described herein may be a cleavable lipid.Non-limiting examples of cleavable lipids include HGT4001, HGT4002,HGT4003, HGT4004 and/or HGT4005.

The neutral lipids may have any suitable molecular weight. In certainembodiments, the molecular weight of a neutral lipid may be less than orequal to about 2,500 g/mol, less than or equal to about 2,000 g/mol,less than or equal to about 1,500 g/mol, less than or equal to about1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equalto about 900 g/mol, less than or equal to about 800 g/mol, less than orequal to about 700 g/mol, less than or equal to about 600 g/mol, lessthan or equal to about 500 g/mol, less than or equal to about 400 g/mol,less than or equal to about 300 g/mol, less than or equal to about 200g/mol, or less than or equal to about 100 g/mol. In some instances, themolecular weight of a neutral lipid may be greater than or equal toabout 100 g/mol, greater than or equal to about 200 g/mol, greater thanor equal to about 300 g/mol, greater than or equal to about 400 g/mol,greater than or equal to about 500 g/mol, greater than or equal to about600 g/mol, greater than or equal to about 700 g/mol, greater than orequal to about 1000 g/mol, greater than or equal to about 1,250 g/mol,greater than or equal to about 1,500 g/mol, greater than or equal toabout 1,750 g/mol, greater than or equal to about 2,000 g/mol, orgreater than or equal to about 2,250 g/mol. Combinations of the aboveranges (e.g., at least about 200 g/mol and less than or equal to about2,500 g/mol) are also possible. In embodiments in which more than onetype of neutral lipid are present in a particle, each type of neutrallipid may independently have a molecular weight in one or more of theranges described above.

In some embodiments, the percentage (e.g., by weight, or by mole) of asingle type of neutral lipid and/or of all the neutral lipids in aparticle may be greater than or equal to about 0.5%, greater than orequal to about 1%, greater than or equal to about 2%, greater than orequal to about 4%, greater than or equal to about 6%, greater than orequal to about 8%, greater than or equal to about 10%, greater than orequal to about 15%, or greater than or equal to about 20%. In someinstances, the percentage (e.g., by weight, or by mole) may be less thanor equal to about 20%, less than or equal to about 18%, less than orequal to about 15%, less than or equal to about 412%, less than or equalto about 10%, less than or equal to about 8%, less than or equal toabout 6%, less than or equal to about 5%, less than or equal to about4%, or less than or equal to about 3%. Combinations of the abovereferenced ranges are also possible (e.g., greater than or equal to 0.5%and less than or equal to about 20%). The percentage (e.g., by weight,or by mole) may be determined as described above with respect toionizable molecules. In embodiments in which more than one type ofneutral lipid are present in a particle, each type of neutral lipid mayindependently have a percentage (e.g., by weight, or by mole) withrespect to the particle in one or more of the ranges described above.

In some embodiments, the particle comprises one or more moleculescapable of reducing particle aggregation. Non-limiting examples ofmolecules capable of reducing particle aggregation includePEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 orC14-PEG), PEG-cDMA, PEG-DSG (1,2-Distearoyl-sn-glycerol,methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol), andPEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). Othermolecules are also possible.

The molecule capable of reducing particle aggregation may have anysuitable molecular weight. In certain embodiments, the molecular weightof a molecule capable of reducing particle aggregation may be less thanor equal to about 2,500 g/mol, less than or equal to about 2,000 g/mol,less than or equal to about 1,500 g/mol, less than or equal to about1,250 g/mol, less than or equal to about 1,000 g/mol, less than or equalto about 900 g/mol, less than or equal to about 800 g/mol, less than orequal to about 700 g/mol, less than or equal to about 600 g/mol, lessthan or equal to about 500 g/mol, less than or equal to about 400 g/mol,less than or equal to about 300 g/mol, less than or equal to about 200g/mol, or less than or equal to about 100 g/mol. In some instances, themolecular weight of a molecule capable of reducing particle aggregationmay be greater than or equal to about 100 g/mol, greater than or equalto about 200 g/mol, greater than or equal to about 300 g/mol, greaterthan or equal to about 400 g/mol, greater than or equal to about 500g/mol, greater than or equal to about 600 g/mol, greater than or equalto about 700 g/mol, greater than or equal to about 1000 g/mol, greaterthan or equal to about 1,250 g/mol, greater than or equal to about 1,500g/mol, greater than or equal to about 1,750 g/mol, greater than or equalto about 2,000 g/mol, or greater than or equal to about 2,250 g/mol.Combinations of the above ranges (e.g., at least about 200 g/mol andless than or equal to about 2,500 g/mol) are also possible. Inembodiments in which more than one type of molecule capable of reducingparticle aggregation are present in a particle, each type of moleculemay independently have a molecular weight in one or more of the rangesdescribed above.

In some embodiments, the percentage (e.g., by weight, or by mole) of asingle type of molecule capable of reducing particle aggregation and/orof all the molecules capable of reducing particle aggregation in aparticle may be greater than or equal to about 0.5%, greater than orequal to about 1%, greater than or equal to about 2%, greater than orequal to about 4%, greater than or equal to about 6%, greater than orequal to about 8%, greater than or equal to about 10%, greater than orequal to about 12%, greater than or equal to about 15%, or greater thanor equal to about 18%. In some instances, the percentage (e.g., byweight, or by mole) may be less than or equal to about 20%, less than orequal to about 18%, less than or equal to about 15%, less than or equalto about 12%, less than or equal to about 10%, less than or equal toabout 8%, less than or equal to about 7%, less than or equal to about6%, less than or equal to about 5%, or less than or equal to about 4%.Combinations of the above referenced ranges are also possible (e.g.,greater than or equal to 0.5% and less than or equal to about 7%,greater than or equal to 2% and less than or equal to about 20%). Inembodiments in which more than one type of molecule capable of reducingparticle aggregation are present in a particle, each type of moleculemay independently have a percentage (e.g., by weight, or by mole) withrespect to the particle in one or more of the ranges described above.The percentage (e.g., by weight, or by mole) may be determined asdescribed above with respect to ionizable molecules.

In some embodiments, the particles may comprise a reactive componentattached to dienophile as a chemically cleavable group which mayprovoking the release in vitro of the formulation and/or thepolynucleotide.

In some embodiments, the particles may comprise a polymer, such aspolyethylenimine (PEI), dithiobis(succinimidylpropionate) (DSP),Dimethyl-3,3′-dithiobispropionimidate (DTBP), poly(ethylene imine)biscarbamate (PEIC), poly(L-lysine) (PLL), histidine modified PLL,poly(N-vinylpyrrolidone) (PVP), poly(propylenimine (PPI),poly(amidoamine) (PAMAM), poly(amido ethylenimine) (SS-PAEI),triehtylenetetramine (TETA), poly(β-aminoester), poly(4-hydroxy-L-proineester) (PHP), poly(allylamine), poly(α-[4-aminobutyl]-L-glycolic acid(PAGA), Poly(D,L-lactic-co-glycolid acid (PLGA),Poly(N-ethyl-4-vinylpyridinium bromide), poly(phosphazene)s (PPZ),poly(phosphoester)s (PPE), poly(phosphoramidate)s (PPA),poly(N-2-hydroxypropylmethacrylamide) (pHPMA),poly(2-(dimethylamino)ethyl methacrylate) (pDMAEMA), poly(2-aminoethylpropylene phosphate) PPE_EA), chitsoan, galactosylated chitosan,N-dodecylated chitosan, histone, collagen or dextran-spermine, andcombinations thereof. In one embodiment, the polymer may be an inertpolymer such as, but not limited to, PEG. In one embodiment, the polymermay be a cationic polymer such as, but not limited to, PEI, PLL, TETA,poly(allylamine), Poly(N-ethyl-4-vinylpyridinium bromide), pHPMA andpDMAEMA. In one embodiment, the polymer may be a biodegradable PEI suchas, but not limited to, DSP, DTBP and PEIC. In one embodiment, thepolymer may be biodegradable such as, but not limited to, histinemodified PLL, SS-PAEI, poly(β-aminoester), PHP, PAGA, PLGA, PPZ, PPE,PPA and PPE-EA.

In some embodiments, the particles described herein may be liposomesincluding one or more polynucleotides. In some cases, liposomes mayencapsulate (e.g., partially, completely) the one or morepolynucleotides.

In certain embodiments, a particle, formulation, or compositiondescribed herein has components and/or a configuration as described inInternational Pub. No. WO2013/090648, entitled “Modified nucleoside,nucleotide, and nucleic acid compositions”, filed Dec. 14, 2012, and/orU.S. Pub. No. US2012/0295832, entitled “Novel Lipids and Compositionsfor Intracellular Delivery of Biologically Active Compounds”, filed May8, 2012, each of which is incorporated herein by reference in itsentirety for all purposes.

In some embodiments, the ratio of one or more components (e.g., lipids,all other components) to polynucleotides (e.g., mRNA or alternative ormodified mRNA alternative or modified mRNA) in the particles may begreater than or equal to about 5:1, greater than or equal to about 10:1,greater than or equal to about 15:1, greater than or equal to about20:1, greater than or equal to about 25:1, greater than or equal toabout 30:1, greater than or equal to about 35:1, greater than or equalto about 40:1, greater than or equal to about 45:1, greater than orequal to about 50:1, greater than or equal to about 55:1, or greaterthan or equal to about 60:1. In some embodiments, the ratio of one ormore components to polynucleotides may be less than or equal to about70:1, less than or equal to about 65:1, less than or equal to about60:1, less than or equal to about 55:1, less than or equal to about50:1, less than or equal to about 45:1, less than or equal to about40:1, less than or equal to about 35:1, less than or equal to about30:1, less than or equal to about 25:1, less than or equal to about20:1, less than or equal to about 15:1, or less than or equal to about10:1 Combinations of the above referenced ranges are also possible(e.g., greater than or equal to 1:1 and less than or equal to about20:1). Other ranges are also possible.

In some embodiments, the particles comprise a lipid, in particular, anionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In one set of embodiments, a particle formulation (e.g., lipidnanoparticle formulation) consists essentially of (i) at least one lipidselected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate(L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE andSM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g.,PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

In one set of embodiments, a formulation includes from about 25% toabout 75% on a molar basis of a cationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., fromabout 35 to about 65%, from about 45 to about 65%, about 60%, about57.5%, about 50% or about 40% on a molar basis.

In one set of embodiments, a formulation includes from about 0.5% toabout 15% on a molar basis of a neutral lipid e.g., from about 3 toabout 12%, from about 5 to about 10% or about 15%, about 10%, or about7.5% on a molar basis. Exemplary neutral lipids include, but are notlimited to, DSPC, POPC, DPPC, DOPE and SM. In one set of embodiments,the formulation includes from about 5% to about 50% on a molar basis ofa sterol (e.g., about 15 to about 45%, about 20 to about 40%, about 40%,about 38.5%, about 35%, or about 31% on a molar basis. An exemplarysterol is cholesterol. In one set of embodiments, the formulationincludes from about 0.5% to about 20% on a molar basis of the PEG orPEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to about 5%,about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molarbasis. In one set of embodiments, the PEG or PEG modified lipidcomprises a PEG molecule of an average molecular weight of 2,000 Da. Inother embodiments, the PEG or PEG modified lipid comprises a PEGmolecule of an average molecular weight of less than 2,000, for examplearound 1,500 Da, around 1,000 Da, or around 500 Da. ExemplaryPEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG),PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107,276-287 (2005).)

In one set of embodiments, a formulations includes 25-75% of a cationiclipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% of aneutral lipid, 5-50% of a sterol, and 0.5-20% of a PEG or PEG-modifiedlipid on a molar basis.

In one set of embodiments, a formulation includes 35-65% of a cationiclipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% of aneutral lipid, 15-45% of a sterol, and 0.5-10% of a PEG or PEG-modifiedlipid on a molar basis.

In one set of embodiments, a formulation includes 45-65% of a cationiclipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% of aneutral lipid, 25-40% of a sterol, and 0.5-10% of a PEG or PEG-modifiedlipid on a molar basis.

In one set of embodiments, a formulation includes about 60% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5%of a neutral lipid, about 31% of a sterol, and about 1.5% of a PEG orPEG-modified lipid on a molar basis.

In one set of embodiments, a formulation includes about 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofa neutral lipid, about 38.5% of a sterol, and about 1.5% of a PEG orPEG-modified lipid on a molar basis.

In one set of embodiments, a formulation includes about 50% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% ofa neutral lipid, about 35% of a sterol, about 4.5% or about 5% of a PEGor PEG-modified lipid, and about 0.5% of a targeting lipid on a molarbasis. Non-limiting examples of a targeting lipid include lipids thatare conjugated to a peptide, a small molecule, an antibody, an aptamer,and/or or fragment protein.

In one set of embodiments, a formulations includes about 40% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate(L319), about 15% of a neutral lipid, about 40% of a sterol, and about5% of a PEG or PEG-modified lipid on a molar basis.

In one set of embodiments, a formulation includes about 57.2% of acationic lipid selected from2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate(L319), about 7.1% of a neutral lipid, about 34.3% of a sterol, andabout 1.4% of a PEG or PEG-modified lipid on a molar basis.

In one set of embodiments, a formulation includes about 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (1. Controlled Release, 107, 276-287(2005)), about 7.5% of a neutral lipid, about 31.5% of a sterol, andabout 3.5% of a PEG or PEG-modified lipid on a molar basis.

In one set of embodiments, a formulation consists essentially of a lipidmixture in molar ratios of about 20-70% cationic lipid: 5-45% neutrallipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. For instance, theformulation may have in a molar ratio of about 20-60% cationic lipid:5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid. Insome such cases, the formulation includes (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), DSPC,cholesterol, and PEG2000-DMG.

In particular embodiments, the molar lipid ratio is approximately50/10/38.5/1.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG, PEG-DSG or PEG-DPG),57.2/7.1134.3/1.4 (mol % cationic lipid/neutral lipid, e.g.,DPPC/Chol/PEG-modified lipid, e.g., PEG-cDMA), 40/15/40/5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG), 50/10/35/4.5/0.5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol PEG-modified lipid, e.g., PEG-DSG), 50/10/35/5 (cationiclipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG),40/10/40/10 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA), 35/15/40/10(mol % cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid,e.g., PEG-DMG or PEG-cDMA) or 52/13/30/5 (mol % cationic lipid/neutrallipid, e.g., DSPC/Chol PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Exemplary formulations (e.g., lipid nanoparticle compositions) andmethods of making the same are described, for example, in Semple et al.(2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem.Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21,1570-1578 (each of which is incorporated herein by reference).

In some cases, the particles in the filtered suspension and/or theparticles of a composition/formulation described herein may have arelatively narrow distribution in a cross-sectional dimension. Forinstance, in certain embodiments, the coefficient of variation of across-sectional dimension (e.g., diameter) of the particles in asuspension and/or composition/formulation may be less than or equal toabout 30%, less than or equal to about 25%, less than or equal to about20%, less than or equal to about 15%, less than or equal to about 10%,or less than or equal to about 5%. In certain embodiments, thecoefficient of variation of a cross-sectional dimension (e.g., diameter)of the particles in a suspension and/or composition/formulation may begreater than or equal to about 1%, greater than or equal to about 3%,greater than or equal to about 5%, greater than or equal to about 7%, orgreater than or equal to about 10%. Combinations of the above-referencedranges are also possible (e.g., greater than or equal to about 1% andless than or equal to about 20%, greater than or equal to about 5% andless than or equal to about 20%).

In some embodiments, the particles in the filtered suspension and/or theparticles of a composition/formulation described herein may have arelatively small average cross-sectional dimension (e.g., diameter). Forinstance, in some embodiments, the average cross-sectional dimension(e.g., average diameter) of the particles in a filtered suspension orcomposition/formulation may be less than or equal to about 1,000 nm,less than or equal to about 800 nm, less than or equal to about 600 nm,less than or equal to about 500 nm, less than or equal to about 400 nm,less than or equal to about 300 nm, less than or equal to about 200 nm,less than or equal to about 150 nm, less than or equal to about 120 nm,less than or equal to about 100 nm, or less than or equal to about 50nm. In some embodiments, the average cross-sectional dimension (e.g.,average diameter) of the particles in a filtered suspension orcomposition/formulation may be greater than or equal to about 10 nm,greater than or equal to about 50 nm, greater than or equal to about 70nm, greater than or equal to about 100 nm, greater than or equal toabout 200 nm, greater than or equal to about 300 nm, greater than orequal to about 400 nm, greater than or equal to about 500 nm, greaterthan or equal to about 600 nm, greater than or equal to about 700 nm,greater than or equal to about 800 nm, or greater than or equal to about900 nm. combinations of the above-referenced ranges are also possible(e.g., greater than or equal to about 50 nm and less than or equal toabout 200 nm, greater than or equal to about 70 nm and less than orequal to about 120 nm).

As used herein, the diameter of a particle for a non-spherical particleis the diameter of a perfect mathematical sphere having the same volumeas the non-spherical particle. In general, the particles describedherein are approximately spherical; however the particles are notnecessarily spherical but may assume other shapes (e.g., discs, rods) aswell.

In some embodiments, the particles are microparticles. In certainembodiments, the particles may have an average cross-sectional dimensionof less than 1 mm. For instance, in some embodiments, the averagecross-sectional dimension of the particles may be less than or equal toabout 1,000 microns, less than or equal to about 500 microns, less thanor equal to about 100 microns, less than or equal to about 50 microns,less than or equal to about 10 microns, or less than or equal to about 5microns. In some embodiments, the average cross-sectional dimension ofthe particles may be greater than or equal to about 1 micron.Combinations of the above-referenced ranges are also possible.

In some embodiments, the particles are biocompatible. As used herein,the term “biocompatible” is intended to describe a material (e.g.,particles, excipients) that is not toxic to cells. Particles are“biocompatible” if their addition to cells in vitro results in less thanabout 20% (e.g., less than about 15%, less than about 10%, less thanabout 5%, less than about 3%, less than about 2%, less than about 1%)cell death, and their administration in vivo does not substantiallyinduce inflammation or other such adverse effects.

In some embodiments, the particles are biodegradable. As used herein,“biodegradable” compounds are those that, when introduced into cells,are broken down by the cellular machinery or by hydrolysis intocomponents that the cells can either reuse or dispose of withoutsignificant toxic effects on the cells (i.e., less than about 20% (e.g.,less than about 15%, less than about 10%, less than about 5%, less thanabout 3%, less than about 2%, less than about 1%) of the cells arekilled when the components are added to cells in vitro). The componentspreferably do not induce inflammation or other adverse effects in vivo.In certain embodiments, the chemical reactions relied upon to break downthe biodegradable compounds are uncatalyzed. For example, the inventivematerials may be broken down in part by the hydrolysis of the polymericmaterial of the inventive coated particles.

In some embodiments, a method described herein may comprise adding anadditional component to the particle. For instance, in some embodiments,a method may comprise attaching a surface modifying agent to the surfaceof the particle. In general, any suitable chemical compound can beattached to particle. Non-limiting examples of chemical compoundsinclude small molecules, polynucleotides, proteins, peptides, metals,polymers, oligomers, organometallic complexes, lipids, carbohydrates,etc. The chemical compound may modify any property of particle includingsurface charge, hydrophilicity, hydrophobicity, zeta potential, size,etc. In certain embodiments, the chemical compound is a polymer such aspolyethylene glycol (PEG). In certain embodiments, the chemical compoundis a targeting moiety used to direct the particles to a particular cell,collection of cells, tissue, or organ system and/or to promoteendocytosis or phagocytosis of the particle. Any targeting moiety knownin the art of drug delivery may be used.

In some embodiments, the particles described herein may be made in asterile environment.

In some embodiments, the particles may be created using microfluidictechnology (see, e.g., Whitesides, George M., “The Origins and theFuture of Microfluidics”. Nature, 2006 442: 368-373; Stroock et al.,“Chaotic Mixer for Microchannels”. Science, 2002 295: 647-651; andValencia et al., “Microfluidic Platform for Combinatorial Synthesis andOptimization of Targeted Nanoparticles for Cancer Therapy”. ACS Nano2013 (DOI/10.1021/nn403370e). As a non-limiting example, controlledmicrofluidic formulation includes a passive method for mixing streams ofsteady pressure-driven flows in micro channels at a low Reynolds numberas described, e.g., in Stroock et al., “Chaotic Mixer forMicrochannels”. Science, 2002 295: 647-651).

In one embodiment, the particles may be created using a micromixer chipsuch as, but not limited to, those from Harvard Apparatus (Holliston,Mass.) or Dolomite Microfluidics (Royston, UK). A micromixer chip can beused for rapid mixing of two or more fluid streams with a split andrecombine mechanism.

In one embodiment, the particles may be created using NanoAssemblerY-mixer chip technology.

Particles formed via the methods described herein may be particularlyuseful for administering an agent to a subject in need thereof. In someembodiments, the particles are used to deliver a pharmaceutically activeagent. In some instances, the particles are used to deliver aprophylactic agent. The particles may be administered in any way knownin the art of drug delivery, for example, orally, parenterally,intravenously, intramuscularly, subcutaneously, intradermally,transdermally, intrathecally, submucosally, sublingually, rectally,vaginally, etc.

Once the particles have been prepared, they may be combined withpharmaceutically acceptable excipients to form a pharmaceuticalcomposition. As would be appreciated by one of skill in this art, theexcipients may be chosen based on the route of administration asdescribed below, the agent being delivered, and the time course ofdelivery of the agent.

Pharmaceutical compositions described herein and for use in accordancewith the embodiments described herein may include a pharmaceuticallyacceptable excipient. As used herein, the term “pharmaceuticallyacceptable excipient” means a non-toxic, inert solid, semi-solid orliquid filler, diluent, encapsulating material or formulation auxiliaryof any type. Some examples of materials which can serve aspharmaceutically acceptable excipients are sugars such as lactose,glucose, and sucrose; starches such as corn starch and potato starch;cellulose and its derivatives such as sodium carboxymethyl cellulose,methylcellulose, hydroxypropylmethylcellulose, ethyl cellulose, andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipientssuch as cocoa butter and suppository waxes; oils such as peanut oil,cottonseed oil; safflower oil; sesame oil; olive oil; corn oil andsoybean oil; glycols such as propylene glycol; esters such as ethyloleate and ethyl laurate; agar; detergents such as Tween 80; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen free water; isotonic saline; citric acid, acetate salts,Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e., theparticles), the liquid dosage forms may contain inert diluents commonlyused in the art such as, for example, water or other solvents,solubilizing agents and emulsifiers such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3 butylene glycol, dimethylformamide, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, ethanol, U.S.P., and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilcan be employed including synthetic mono or diglycerides. In addition,fatty acids such as oleic acid are used in the preparation ofinjectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration may be suppositorieswhich can be prepared by mixing the particles with suitable nonirritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax which are solid at ambient temperature butliquid at body temperature and therefore melt in the rectum or vaginalcavity and release the particles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of apharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also possible.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the particles in a proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the particles in a polymer matrixor gel.

Kits for use in preparing or administering the particles are alsoprovided. A kit for forming particles may include any solvents,solutions, buffer agents, acids, bases, salts, targeting agent, etc.needed in the particle formation process. Different kits may beavailable for different targeting agents. In certain embodiments, thekit includes materials or reagents for purifying, sizing, and/orcharacterizing the resulting particles. The kit may also includeinstructions on how to use the materials in the kit. The one or moreagents (e.g., pharmaceutically active agent) to be encapsulated in theparticle are typically provided by the user of the kit.

Kits are also provided for using or administering the particles orpharmaceutical compositions thereof. The particles may be provided inconvenient dosage units for administration to a subject. The kit mayinclude multiple dosage units. For example, the kit may include 1-100dosage units. In certain embodiments, the kit includes a week supply ofdosage units, or a month supply of dosage units. In certain embodiments,the kit includes an even longer supply of dosage units. The kits mayalso include devices for administering the particles or a pharmaceuticalcomposition thereof. Exemplary devices include syringes, spoons,measuring devices, etc. The kit may optionally include instructions foradministering the particles (e.g., prescribing information).

“Composition”: The terms “composition” and “formulation” are usedinterchangeably.

“Condition”: As used herein, the terms “condition,” “disease,” and“disorder” are used interchangeably.

The following examples are intended to illustrate certain embodiments ofthe present invention, but do not exemplify the full scope of theinvention.

EXAMPLES Example 1

This example describes the effect of incubation time and pH onencapsulation efficiency of nanoparticles containing mRNA and anionizable molecule (i.e., MC3 lipid). The particles also contained otherlipids and a sterol and are referred to in the examples as lipidnanoparticles. Encapsulation percentages of greater than 90% wereobtained under acidic conditions when incubations times were at least300 seconds under acidic conditions.

Lipid nanoparticles were prepared using a micromixer channel in aT-configuration at sufficient flow rates to facilitate rapid mixing ofan aqueous solution containing mRNA and an ethanol solution containinglipids. The mixing led to nano-precipitation and ultimately particleformation. The lipids used in this experiment were PEG-DMG (a diffusiblePEG lipid which may impart physical stability to the lipidnanoparticle), cholesterol (e.g., which may provide structural supportto the lipid nanoparticle), DSPC (a phospholipid known to be involved inlipid fusogenecity with the endosome compartment of a cell), and MC3 (anionizable cationic lipid that becomes protonated in a low pH endosomalenvironment, which may lead to escape of the endosome compartment to thecytosol).

The ethanol solution was prepared by dissolving MC3, DSPC, cholesterol,and PEG-DMG at a mol % ratio of 50:10:38.5:1.5 in 200 proof ethanol toobtain a final lipid concentration of 12.5 mM. The stock ethanolsolution was then filtered through a 0.8/0.2 micron filter. The ethanolsolution was then stored at room temperature until use.

The aqueous solution contained about 0.2 mg/mL of mRNA in 20 mM sodiumcitrate pH 4.0. A tonicity modifier, sucrose, was added to adjust thetotal osmolarity of the solution. Once prepared, the aqueous solutionwas filtered through a 0.8/0.2 micron filter. The aqueous solution wasthen stored at room temperature until use.

Lipid nanoparticles were assembled by mixing the aqueous mRNA and lipidsolutions using an impinging jet mixer in a Tee configuration. The flowratios were offset between lipid and aqueous solutions, resulting in atotal ethanol content at the site of mixing of 33%. The total cumulativeflow rate of the aqueous and lipid solutions was 125 mL/min. The lipidnanoparticles produced were then diluted in-line at a 1:1 ratio with 20mM sodium citrate pH 6.0 which was then followed by a second in-linedilution at 2:1 ratio with lx citrate buffered saline (CBS). In thisexample, the first dilution was set at a cumulative flow rate of 125mL/min followed by a second dilution at a cumulative flow rate of 250mL/min. The particles were allowed to form for different incubationtimes. The incubation time was measured at the point at which the all ofthe components in the lipid and aqueous solutions were combined andended with removal of the particles in this example.

To investigate incubation times and the influence on percentencapsulation, the pH of the dilution buffers were adjusted to increasesolution pH from less than about 6.5 to greater than about 7.0 at thesite of dilution. The dilution time was modified by changing the lengthof tubing between the mixing Tee and the point of dilution, thusmodulating the residence time within acidic buffer. Percentencapsulation was quantified by the Ribogreen fluorescence assay afterbuffer exchange into PBS via dialysis or TFF FIG. 4 shows a graph ofpercent encapsulation versus incubation time (also referred to asresidence time) in the resulting acidic buffer (pH less than about 6.0)from the combination of the lipid and aqueous solutions. Encapsulationpercentages of greater than 90% were achieved at 300 seconds as shown inFIG. 4 .

Example 2

This example describes the change in surface charge of the nanoparticlesassociated with the altering step, which changes the pH of thesuspension to a pH that is greater than the pKa of the ionizablemolecule in the particles. This example shows that the surface charge oflipid nanoparticles containing ionizable lipid changed when the pH wasaltered to be greater than the pKa of the ionizable lipid. The magnitudeof the change in surface charge was greater for lipid nanoparticlescontaining mRNA than for lipid nanoparticles that did not contain mRNA.

Lipid nanoparticles containing mRNA were formed as described in Example1. Lipid nanoparticles that did not contain mRNA (“Empty”) were formedby a similar process as described in Example 1 except the aqueoussolution did not contain mRNA. The ionizable lipid had a pKa of 6.3.After an incubation time of at least 300 seconds, the pH of thesuspension containing the mRNA and the suspension containing the emptylipid nanoparticles was altered by changing the pH of the suspension.Particles were further purified by TFF and were subsequently filteredthrough a sterilizing-grade membrane. The zeta potential of the lipidparticles were measured at various pHs of the resulting suspension usinga Wyatt Mobius zeta-potentiometer. FIG. 5 shows a graph of zetapotential versus pH for mRNA and empty lipid nanoparticles. As shown inFIG. 5 , the zeta potential of the empty and mRNA lipid nanoparticlesare substantially the same until the isoelectric point of the ionizablelipid. After the isoelectric point, the surface charge of the mRNA lipidnanoparticles were substantially more negative than the emptynanoparticles.

Example 3

This example describes the filtration properties of suspensionscontaining mRNA lipid nanoparticles that have a pH greater than the pKaof the ionizable lipid in the particle. When the pH of the suspensionwas greater than the pKa of the ionizable lipid, the filtration time wasreduced, a higher permeate flux was achieved throughout the filtrationprocess, and the coefficient in variation of the resulting particles wasreduced compared to a suspension having a pH less than the pKa of theionizable lipid (control). Lipid nanoparticles containing mRNA wereformed as described in Example 1.

The pKa of the ionizable lipid was 6.3. After formation of the particlesand allowing the particles to incubate for at least 300 seconds, the pHof the suspension was changed from a pH of about 5.7 to a pH of about7.4 in an altering step. The pH was altered by volumetric addition of a1M Tris pH 8 buffer to a final buffer composition of 100 mM Tris. Thecontrol suspension was also incubated for at least 300 second but the pHof the suspension was not altered and remained at about pH 5.7 prior todiafiltration. However, pH 5.7 buffer was added to the control to ensurethat the concentration of particles were substantially the same in bothsuspension. For the control, the pH of the suspension was changedgradually during filtration using diafiltration. The pH was increasedduring the diafiltration step with each diafiltration volume (DV) and apH of 7.0 was reached after six diafiltration volumes. The pH at two DVand four DV were 6.05 and 6.41, respectively.

Both suspensions were filtered using tangential flow filtration. Thetangential flow filtration process involved a first concentration step,then a diafiltration step using six diafiltration volumes, and a secondconcentration step. FIG. 6A shows the permeate flux versus time for thetwo suspensions. The suspension having a pH of about 7.4 had afiltration time of about 50 minutes while the suspension having a pH ofabout 5.7 has a filtration time of about 140 minutes. The filtrationtime included a first concentration step, diafiltration with 12diafiltration volumes, and a final concentration step. The higherpermeate flux achieved with the suspension having a pH of about 7.4increased the filtration rate resulting in a lower filtration time. FIG.6B shows the permeate flux at each stage of the tangential flowfiltration for both suspensions. As shown in FIG. 6B, the permeate fluxfor the suspension having a pH of about 7.4 was greater than thesuspension having a pH of about 5.7 during all stages of filtration.Thus, the suspension having a pH greater than the pKa of the ionizablelipid had better filtration performance than the suspension having a pHless than the pKa of the ionizable lipid.

The pH of the suspension during tangential flow filtration alsoinfluenced the average particle size, as well as polydispersity, of thefiltered suspension. FIG. 7A shows the average particle size duringvarious stages of tangential flow filtration (wherein DV stands fordiafiltration volume), and after a sterile filtration for bothsuspensions. As shown in FIG. 7A, the average particle size of theparticles in the suspension having a pH of about 7.4 was smaller thanthe average particle size of the particles in the suspension having a pHof about 5.7 after tangential flow filtration and sterile filtration.FIG. 7B shows the percent polydispersity for the particles duringvarious stages of tangential flow filtration and after a sterilefiltration for both suspensions. As shown in FIG. 7B, the suspensionhaving a pH of about 7.4 had a significantly lower polydispersity inaverage particle size than the suspension having a pH of about 5.7.Noteably, after sterile filtration, the pH 7.4 suspension had apolydispersity of less than about 3% and the pH 5.7 suspension had apolydispersity of about 20%.

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1-44. (canceled)
 45. A composition comprising: a plurality of particlescomprising mRNA comprising greater than or equal to 100 nucleotides andless than or equal to 10,000 nucleotides in length and an ionizablemolecule, wherein: an average cross-sectional dimension of the particlesin the composition is less than or equal to about 150 nm, a coefficientof variation of a cross-sectional dimension of the particles in thecomposition is less than or equal to about 20%, and a weight percentageof mRNA in the particles is greater than or equal to about 50% and lessthan or equal to about 99%, and wherein the composition is formed by(ia) changing a pH of a suspension comprising the plurality of particlesfrom a first pH to a second pH, wherein the second pH is greater thanthe pKa of the ionizable molecule, wherein the pKa of the ionizablemolecule is greater than or equal to about 6, or (ib) changing anaverage zeta potential of a suspension comprising the plurality ofparticles, from a first zeta potential to a second zeta potential,wherein the second zeta potential is less than the first zeta potential,and subsequently, (ii) filtering the suspension.
 46. The composition ofclaim 45, wherein the particles further comprise a molecule capable ofreducing particle aggregation.
 47. The composition of claim 46, whereinthe molecule capable of reducing particle aggregation is a PEG lipid ora PEG-modified lipid.
 48. The composition of claim 45, wherein theionizable molecule comprises a nitrogen.
 49. The composition of claim45, wherein the ionizable molecule is a cationic lipid.
 50. Thecomposition of claim 49, wherein the cationic lipid is selected from thegroup consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1.3]-dioxolane,dilinolcyl-methyl-4-dimethylaminobutyrate, and di((Z)-non-2-cn-l-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate.
 51. The compositionof claim 1, wherein the particle comprises a sterol molecule.
 52. Thecomposition of claim 51, wherein the sterol molecule is a cholesterolmolecule.
 53. The composition of claim 45, wherein the filteredsuspension is formed by (ib) and the first zeta potential is greaterthan or equal to about 0 mV.
 54. The composition of claim 45, whereinthe filtered suspension is formed by (ib) and the particles have a zetapotential of less than or equal to about −1 mV.
 55. The composition ofclaim 45, wherein the filtered suspension is formed by (ia) and the pHof the suspension prior to the changing step is less than or equal toabout the pKa of the ionizable molecule.
 56. The composition of claim45, wherein the filtered suspension is formed by (ia) and the pKa ofionizable molecule is between about 6 and
 7. 57. The composition as inclaim 45, wherein the pKa of the ionizablc molecule is about 6.2. 58.The composition of claim 45, wherein a coefficient of variation of across-sectional dimension of the particles in the filtered suspension isless than or equal to about 15%.
 59. The composition of claim 45,wherein the filtering step comprises passing at least a portion of thesuspension thorough a porous substrate.
 60. The composition of claim 45,wherein the filtering step comprises passing the suspension through afilter having a mean pore size of less than or equal to about 0.2microns after the tangential flow filtration.
 61. The composition ofclaim 45, wherein the permeate flux throughout the filtering step isless than or equal to about 120 L/m.sup.2h when the transmembranepressure is between about 1 psi and about 20 psi, or between about 10psi and about 15 psi.
 62. The composition of claim 45, wherein thefiltering step comprises tangential flow filtration (TFF).